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David R Colman

Publications and source records attributed to David R Colman.

16 recordsLinked to original sources
PubMed2006

The CNS synapse revisited: gaps, adhesive welds, and borders.

Although processes leading up to the point of synapse formation are fairly well understood, the precise sequence of events in which the membranes of two separate cells "lock in" to form a mature synaptic junctional complex is poorly understood. A careful study of the molecules operating at the synapse indicates that their roles are more multifarious than once imagined. In this review we posit that the synapse is a functional organelle with poorly defined boundaries and a complex biochemistry. The role of adhesion molecules, including the integration of their signaling and adhesive properties in the context of synaptic activity is discussed.

Animals↗
PubMed2006

A role for Nr-CAM in the patterning of binocular visual pathways.

Retinal ganglion cell (RGC) axons diverge within the optic chiasm to project to opposite sides of the brain. In mouse, contralateral RGCs are distributed throughout the retina, whereas ipsilateral RGCs are restricted to the ventrotemporal crescent (VTC). While repulsive guidance mechanisms play a major role in the formation of the ipsilateral projection, little is known about the contribution of growth-promoting interactions to the formation of binocular visual projections. Here, we show that the cell adhesion molecule Nr-CAM is expressed by RGCs that project contralaterally and is critical for the guidance of late-born RGCs within the VTC. Blocking Nr-CAM function causes an increase in the size of the ipsilateral projection and reduces neurite outgrowth on chiasm cells in an age- and region-specific manner. Finally, we demonstrate that EphB1/ephrin-B2-mediated repulsion and Nr-CAM-mediated attraction comprise distinct molecular programs that each contributes to the proper formation of binocular visual pathways.

Animals↗
PubMed2005

Glial membranes at the node of Ranvier prevent neurite outgrowth.

Nodes of Ranvier are regularly placed, nonmyelinated axon segments along myelinated nerves. Here we show that nodal membranes isolated from the central nervous system (CNS) of mammals restricted neurite outgrowth of cultured neurons. Proteomic analysis of these membranes revealed several inhibitors of neurite outgrowth, including the oligodendrocyte myelin glycoprotein (OMgp). In rat spinal cord, OMgp was not localized to compact myelin, as previously thought, but to oligodendroglia-like cells, whose processes converge to form a ring that completely encircles the nodes. In OMgp-null mice, CNS nodes were abnormally wide and collateral sprouting was observed. Nodal ensheathment in the CNS may stabilize the node and prevent axonal sprouting.

Animals↗
PubMed2005

Proteomic comparison of two fractions derived from the transsynaptic scaffold.

A fraction derived from presynaptic specializations (presynaptic particle fraction; PPF) can be separated from postsynaptic densities (PSD) by adjusting the pH of Triton X-100 (TX-100) extraction of isolated transsynaptic scaffolds. Solubilization of the PPF corresponds to disruption of the presynaptic specialization. We show that the PPF is insoluble to repeated TX-100 extraction at pH 6.0 but becomes soluble in detergent at pH 8.0. By immunolocalization, we find that the major proteins of the PPF, clathrin and dynamin, are concentrated in the presynaptic compartment. By using multidimensional protein identification technology, we compared the protein compositions of the PPF and the PSD fraction. We identified a total of 341 proteins, 50 of which were uniquely found in the PPF, 231 in the PSD fraction, and 60 in both fractions. Comparison of the two fractions revealed a relatively low proportion of actin and associated proteins and a high proportion of vesicle or intracellular compartment proteins in the PPF. We conclude that the PPF consists of presynaptic proteins not connected to the actin-based synaptic framework; its insolubility in pH 6 and solubility in pH 8 buffered detergent suggests that clathrin might be an anchorage scaffold for many proteins in the PPF.

Animals↗
PubMed2005

Differential expression of individual gamma-protocadherins during mouse brain development.

Three tandemly arrayed protocadherin gene clusters (Pcdh-alpha, -beta, -gamma) comprising more than 50 genes are found in human and mouse. Here, we have investigated the expression and distribution of individual gamma-protocadherins (Pcdhs-gamma) in the developing mouse brain. We find that transfection of Pcdh-gamma genes promotes calcium-dependent cell adhesion in HEK 293 cells. Furthermore, Pcdh-gamma can be recruited to synapses of transfected primary hippocampal neurons. Several individual members of the in total 22 Pcdhs-gamma were chosen to examine the expression of the three subfamilies, Pcdh-gammaA, -gammaB, and -gammaC. These Pcdh-gamma transcripts are expressed all over the brain, with minor regional and cell-type specific differences. Interestingly, a distinct, later onset of expression is observed for Pcdh-gammaC5, a gene located at the end of the Pcdh-gamma cluster. Largely overlapping expression patterns of individual Pcdh-gamma proteins are detected with anti-peptide antibodies. Small differences are observed in the staining of dendritic processes and synapse-rich layers. Our results support the idea that Pcdhs-gamma participate in neuronal differentiation and may be implicated in the fine-tuning of neuronal morphology and synaptogenesis. Cell autonomous regulation of transcription might generate the widespread distribution of individual Pcdhs-gamma in the brain, which is strikingly different from the restricted expression patterns observed for classical cadherins. Thus, a defined set of Pcdhs-gamma may engage in neuronal adhesion and signaling on the cellular level.

Animals↗
PubMed2004

Cadherin activity is required for activity-induced spine remodeling.

Neural activity induces the remodeling of pre- and postsynaptic membranes, which maintain their apposition through cell adhesion molecules. Among them, N-cadherin is redistributed, undergoes activity-dependent conformational changes, and is required for synaptic plasticity. Here, we show that depolarization induces the enlargement of the width of spine head, and that cadherin activity is essential for this synaptic rearrangement. Dendritic spines visualized with green fluorescent protein in hippocampal neurons showed an expansion by the activation of AMPA receptor, so that the synaptic apposition zone may be expanded. N-cadherin-venus fusion protein laterally dispersed along the expanding spine head. Overexpression of dominant-negative forms of N-cadherin resulted in the abrogation of the spine expansion. Inhibition of actin polymerization with cytochalasin D abolished the spine expansion. Together, our data suggest that cadherin-based adhesion machinery coupled with the actin-cytoskeleton is critical for the remodeling of synaptic apposition zone.

Actins↗
PubMed2004

The minimal essential unit for cadherin-mediated intercellular adhesion comprises extracellular domains 1 and 2.

N-cadherin comprises five homologous extracellular domains, a transmembrane, and a cytoplasmic domain. The extracellular domains of N-cadherin play important roles in homophilic cell adhesion, but the contribution of each domain to this phenomenon has not been fully evaluated. In particular, the following questions remain unanswered: what is the minimal domain combination that can generate cell adhesion, how is domain organization related to adhesive strength, and does the cytoplasmic domain serve to facilitate extracellular domain interaction? To address these issues, we made serial constructs of the extracellular domains of N-cadherin and produced various cell lines to examine adhesion properties. We show that the first domain of N-cadherin alone on the cell surface fails to generate adhesive activity and that the first two domains of N-cadherin form the "minimal essential unit" to mediate cell adhesion. Cell lines expressing longer extracellular domains or N-cadherin wild type cells formed larger cellular aggregates than those expressing shorter aggregates. However, adhesion strength, as measured by a shearing test, did not reveal any differences among these aggregative cell lines, suggesting that the first two domains of N-cadherin cells generate the same strength of adhesive activity as longer extracellular domain cells. Furthermore, truncations of the first two domains of N-cadherin are also sufficient to form cisdimerization at an adhesive junction. Our findings suggest that the extracellular domains of N-cadherin have distinct roles in cell adhesion, i.e. the first two domains are responsible for homophilic adhesion activity, and the other domains promote adhesion efficiency most likely by positioning essential domains relatively far out from the cell surface.

Animals↗
PubMed2004

Actin-binding proteins in a postsynaptic preparation: Lasp-1 is a component of central nervous system synapses and dendritic spines.

CNS synapses are complex sites of cell-cell communication. Identification and characterization of the protein components of synapses will lead to a better understanding of the mechanisms of neurotransmission and plasticity. We applied multidimensional protein identification technology (MudPIT) to purified, guanidine-solubilized postsynaptic fractions to identify novel synaptically localized molecules. We identified several actin-associated proteins known to regulate actin polymerization and control cell motility in nonneural cells that have not previously been associated with CNS synaptic function. One of these is lasp-1, an actin-associated LIM and SH3 domain-containing protein. We show that lasp-1 is strongly expressed by CNS neurons and is concentrated at synaptic sites. Overall, the preponderance of actin-associated proteins in postsynaptic density fractions, and specifically those involved in actin reorganization, suggests that there are many modes by which the state of synaptic F-actin polymerization and, hence, synaptic physiology are affected.

Actins↗
PubMed2004

Obedient and wayward synaptic behavior.

Synapse formation is a complex process that culminates in the linking up and locking in of pre- and postsynaptic membranes. Shen at al. (2004 [this issue of Cell]) begin to dissect the molecular instructions that govern target selection of pre- and postsynaptic membrane interactions.

Animals↗
PubMed2004

Structure of the neural (N-) cadherin prodomain reveals a cadherin extracellular domain-like fold without adhesive characteristics.

Classical cadherins mediate cell-cell adhesion through calcium-dependent homophilic interactions and are activated through cleavage of a prosequence in the late Golgi. We present here the first three-dimensional structure of a classical cadherin prosequence, solved by NMR. The prototypic prosequence of N-cadherin consists of an Ig-like domain and an unstructured C-terminal region. The folded part of the prosequence-termed prodomain-has a striking structural resemblance to cadherin "adhesive" domains that could not have been predicted from the amino acid sequence due to low sequence similarities. Our detailed structural and evolutionary analysis revealed that prodomains are distant relatives of cadherin "adhesive" domains but lack all the features known to be important for cadherin-cadherin interactions. The presence of an additional "nonadhesive" domain seems to make it impossible to engage homophilic interactions between cadherins that are necessary to activate adhesion, thus explaining the inactive state of prodomain-bearing cadherins.

Amino Acid Sequence↗
PubMed2003

Gamma-protocadherins are targeted to subsets of synapses and intracellular organelles in neurons.

The clustered protocadherins (Pcdhs) comprise >50 putative synaptic recognition molecules that are related to classical cadherins and highly expressed in the nervous system. Pcdhs are organized into three gene clusters (alpha, beta, and gamma). Within the alpha and gamma clusters, three exons encode the cytoplasmic domain for each Pcdh, making these domains identical within a cluster. Using an antibody to the Pcdh-gamma constant cytoplasmic domain, we find that all interneurons in cultured hippocampal neurons express high levels of Pcdh-gamma(s) in a nonsynaptic distribution. In contrast, only 48% of pyramidal-like cells expressed appreciable levels of these molecules. In these cells, Pcdh-gamma(s) were associated with a subset of excitatory synapses in which they may mediate presynaptic to postsynaptic recognition in concert with classical cadherins. Immunogold localization in hippocampal tissue showed Pcdh-gamma(s) at some synapses, in nonsynaptic plasma membranes, and in axonal and dendritic tubulovesicular structures, indicating that they may be exchanged among synapses and intracellular compartments. Our results show that although Pcdh-gamma(s) can be synaptic molecules, synapses form lacking Pcdh-gamma(s). Thus, Pcdh-gamma(s) and their relatives may be late additions to the classical cadherin-based synaptic adhesive scaffold; their presence in intracellular compartments suggests a role in modifying synaptic physiology or stability.

Alternative Splicing↗
PubMed2003

Rapid method for culturing embryonic neuron-glial cell cocultures.

A streamlined, simple technique for primary cell culture from E17 rat tissue is presented. In an attempt to standardize culturing methods for all neuronal cell types in the embryo, we evaluated a commercial medium without serum and used similar times for trypsinization and tested different surfaces for plating. In 1 day, using one method and a single medium, it is possible to produce robust E17 cultures of dorsal root ganglia (DRG), cerebellum, and enteric plexi. Allowing the endogenous glial cells to repopulate the cultures saves time compared with existing techniques, in which glial cells are added to cultures first treated with antimitotic agents. It also ensures that all the cells present in vivo will be present in the culture. Myelination commences after approximately 2 weeks in culture for dissociated DRG and 3-4 weeks in cerebellar cultures. In enteric cultures, glial wrapping of the enteric neurons is seen after 3 weeks (2 weeks in ascorbic acid), suggesting that basal lamina production is important even for glial ensheathment in the enteric nervous system. No overgrowth of fibroblasts or other nonneuronal cells was noted in any cultures, and myelination of the peripheral nervous system and central nervous system cultures was very robust.

Animals↗
PubMed2002

Neural and immunological synaptic relations.

A synapse is a stable adhesive junction between two cells across which information is relayed by directed secretion. The nervous system and immune system utilize these specialized cell surface contacts to directly convey and transduce highly controlled secretory signals between their constituent cell populations. Each of these synaptic types is built around a microdomain structure comprising central active zones of exocytosis and endocytosis encircled by adhesion domains. Surface molecules that may be incorporated into and around the active zones contribute to modulation of the functional state of the synapse.

Animals↗
PubMed2002

Neural (N-) cadherin, a synaptic adhesion molecule, is induced in hippocampal mossy fiber axonal sprouts by seizure.

Aberrant mossy fiber sprouting and synaptic reorganization are plastic responses in human temporal lobe epilepsy, and in pilocarpine-induced epilepsy in rodents. Although the morphological features of the hippocampal epileptic reaction have been well documented, the molecular mechanisms underlying these structural changes are not understood. The classic cadherins, calcium-dependent cell adhesion molecules, are known to function in development in neurite outgrowth, synapse formation, and stabilization. In pilocarpine-induced status epilepticus, the expression of N-cadherin mRNA was sharply upregulated and reached a maximum level (1- to 2.5-fold) at 1- to 4 weeks postseizure in the granule cell layer and the pyramidal cell layer of CA3. N-cadherin protein was correspondingly increased and became concentrated in the inner molecular layer of the dentate gyrus, consistent with the position of mossy fiber axonal sprouts. Moreover, N-cadherin labeling was punctate; colocalized with definitive synaptic markers, and partially localized on polysialated forms of neural cell adhesion molecule (PSA-NCAM)-positive dendrites of granule cells in the inner molecular layer. Our findings show that N-cadherin is likely to be a key factor in responsive synaptogenesis following status epilepticus, where it functions as a mediator of de novo synapse formation.

Animals↗